Membrane-bound antiviral antibodies as receptors for Sendai virions in receptor-depleted erythrocytes

Anti-Sendai virus antibodies were covalently coupled to neuraminidase-treated human erythrocytes by the use of the bifunctional crosslinking reagents, N-succinimidyl-3-(2-pyridyldithio)propionate or succinimidyl-4-(p-maleimidophenyl)butyrate. Neuraminidase-treated erythrocytes bearing antibodies were able to bind Sendai virus particles, while treated erythrocytes lacking the antibodies failed to do so. Virus particles attached to erythrocyte membranes via the antibodies were able to cause hemolysis (virus-cell fusion) and promoted cell-cell fusion. Similar results were obtained when the antibodies were coupled to cat erythrocytes which lack receptors for Sendai virus particles. Reconstituted Sendai virus envelopes, similar to intact virus particles, were able to hemolyze and to induce fusion of neuraminidase-treated antibody-bearing erythrocytes. However, reconstituted envelopes containing inactive HN (hemagglutinin-neuraminidase) but active F (fusion) glycoproteins, despite attachment to antibody-bearing erythrocytes, failed to hemolyze or to induce cell-to-cell fusion. Fusion could be restored by insertion of an active HN glycoprotein into the membranes of the reconstituted envelopes. These results suggest that the HN glycoprotein, besides being the viral attachment protein, also participates in the membrane fusion process.     

The effects of virus and host genes on recombination among ultraviolet-irradiated bacteriophage T4.

The influence of the polA, uvrA, and recA genes of Escherichia coli on recombination among ultraviolet-irradiated T4 bacteriophages was determined with respect to recombination between rII markers and phage yield. The polA and uvrA gene products have no effect on these two aspects of phage DNA metabolism. A recA mutation does not significantly alter rII recombination frequencies in irradiated phage crosses, nor does it greatly change the yield of infectious particles in wild-type phage crosses or crosses in which the phage strains possess the v mutation. However, the same cross experiment performed with a pair of T4x mutants in a recA host demonstrates an 84% reduction in the phage yield in an unirradiated control cross. Furthermore, with increasing doses of uv irradiation, phage productivity of the T4x mutant declines at an accelerated rate compared to T4x⁺ strains crossed in recA cells. Multiplicity reactivation experiments in which wild-type or recombination-deficient (x or y) T4 phages infect wild-type or recombination-deficient (recA) host cells show that irradiated phages can only be reactivated in recA⁺ hosts, regardless of the bacteriophage genotype. These results indicate the involvement of the E. coli recA gene product in normal T4 replication and multiplicity reactivation.     

Biological properties of a hemagglutinin mutant of influenza virus selected by host cells

Chick embryo fibroblast (CEF)-grown stocks of the WSN strain of influenza A(HINI) contain two variants which were designated F and C for fuzzy and clear plaque morphology on Madin-Darby bovine kidney (MDBK) cells. During growth in MDBK cells plaque-isolated F virus was completely replaced by C virus (L. Noronha-Blob and I. T. Schulze (1976), Virology69, 314–322). The parental (F) and the mutant (C) viruses contain hemagglutinins which differ in their ability to bind to host cells. In addition, the host cells from which the purified viruses are obtained affect their binding properties. Thus, as compared to MDBK-grown F virus (F_BK), MDBK-grown C virus (C_BK) produced high amounts of mRNA and high virus yields in MDBK cells. C_BK had greater affinity for SAα2,3Gα1 and SAα2,6Ga1 linkages on derivatized human erythrocytes than did F_BK, independent of whether neuraminidase was present on the virions. C_BK was also resistant to components of calf serum which inhibited F_BK hemagglutination at 37°. As compared to F_BK, C_BK had increased ability to bind to both MDBK cells and CEF at 37° in the presence or absence of an inhibitor of neuraminidase. In addition, when cells with virus bound at 0° were transferred to 37°, C_BK remained cell associated whereas about 80% of FBK dissociated from both cells. Thus, mutation from F to C increased the ability of the virus to associate with MDBK cell receptors. Studies carried out with F and C viruses from both cells indicated that the expression of the mutation depended in part on the host cells in which the virus was grown and in part on the cells used to measure the binding properties. A model relating these observations to selection of HA variants in nature is presented.     

Biochemical studies on influenza viruses. II. Assignment of gene functions to RNA segments 5, 7, and 8 of fowl plague virus and virus N

Two recombinant strains carrying all genes from fowl plague virus (FPV), with the exception of RNA segments 5 and 8 which are derived from virus N, have been characterized by comparative RNA and protein analysis. From tryptic peptide mapping it was ascertained that segment 5 codes for the nucleoprotein, segment 7 for the matrix protein, and segment 8 for the nonstructural protein. In connection with previously published results (Scholtissek et al., 1976; Rohde et al., 1977), these data complete the genetic maps for FPV and virus N.     

Neuraminic acid is involved in the binding of influenza C virus to erythrocytes

Neuraminidases of both viral and bacterial origin have been reported to be unable to destroy the cellular receptor for influenza C virus on chicken erythrocytes, in contrast to the receptors for influenza A and B virus. However, under appropriate conditions neuraminidases from both Vibrio cholerae and Clostridium perfringens were able (i) to make chicken red blood cells resistant against agglutination by influenza C virus and (ii) to reduce the hemagglutination-inhibiting activity of rat serum. Both effects were abolished in the presence of the neuraminidase inhibitor 2,3-dehydro-2-deoxyneuraminic acid (DDN). These results indicate that contrary to previous assumptions sialic acid may very well be an essential component of the receptor for influenza C virus.     

Characterization of host cell binding variants of influenza virus by monoclonal antibodies

We have previously shown that a plaque-type mutant of influenza virus A/WSN has a growth advantage in MDBK cells because its hemagglutinin (HA) has a greater affinity for host cell receptors than does the HA of the parent virus. We show here that the mutant is also less sensitive than the parent to neutralization by antibodies to epitopes in at least two regions on the HA. WSN-specific monoclonal antibodies which had higher radioimmunoassay (RIA) titers against the parent than the mutant virus also had higher plaque inhibition (PI) and hemagglutination inhibition (HI) titers. In contrast, cross-reacting antibodies bound equally well to the parent and mutant viruses as judged by RIA but those which bound to the Cb region of the HA exhibited higher PI and HI titers against the parent virus. The results suggest that preferential neutralization of the parental virus by antibodies can contribute to the selective advantage of mutants which have increased affinity for cellular receptors.     

Characterization of RNA-dependent RNA polymerases in uninfected and cowpea chlorotic mottle virus-infected cowpea leaves: selective removal of host RNA polymerase from membranes containing CCMV RNA replicase.

Extracts from Cowpea chlorotic mottle virus (CCMV)-infected Cowpea leaves contained membrane-bound (31,000 g pellet) and soluble (31,000 g supernatant) RNA-dependent RNA polymerase activities. The membrane-bound RNA-dependent RNA polymerase (CCMV RNA replicase) increased 12-fold 4 days after inoculation. The viral RNA synthesis in vitro proceeded linearly for 20 min and required the four nucleoside triphosphates and Mg²⁺ ions for activity. Manganese ion was a poor substitute for Mg²⁺. Optimal enzymatic activity in vitro was unaffected by exogenous RNA or KCI. The CCMV RNA replicase product was predominantly heterodisperse single-stranded RNAs, some of which comigrated with CCMV virion RNA. Small amounts of large double-stranded RNAs were also products of the replicase reaction. The soluble RNA-dependent RNA polymerase from CCMV-infected or healthy Cowpea leaves required the four nucleoside triphosphates and Mg²⁺ ions for activity. Its activity in the in vitro assay was stimulated by adding exogenous RNAs but was inhibited by KCI. The product of the soluble RNA-dependent RNA polymerase was predominantly double-stranded RNA of approximately 4 to 6 S. RNA-dependent RNA polymerase activity, similar to that detected in the soluble fraction, was detected in the membrane pellet. This activity, which complicates the analysis of viral replicase assay, was removed without affecting CCMV RNA replicase activity by washing the 31,000 g pellets with buffer containing 0.5 M KCI. The KCI treatment aids in preparation of membrane-bound fractions devoid of host RNA-dependent RNA polymerase activity and high in viral replicase activity.     

Cross protection between strains of cucumber mosaic virus: effect of host and type of inoculum on accumulation of virions and double-stranded RNA of the challenge strain

Two strains of cucumber mosaic virus (CMV) differed in three characteristics of value for cross-protection experiments. Their virions and also two of their double-stranded RNAs could be separated and distinguished by electrophoresis on polyacrylamide gels, and the symptoms of one strain were milder than the other in tobacco, tomato, and squash. The mild strain, CMV-S, protected plants of these three hosts from the effects of the second strain, CMV-P, and also prevented the accumulation of virions and ds RNAs of the challenge strain. Protection was detected in leaves inoculated with the challenge strain and also in later formed leaves. The only exception to this result was the accumulation of ds RNAs and to a lesser extent virions of the challenge strain when infectious viral RNA was used as the challenge inoculum instead of virus particles. This breakdown of cross protection occurred only in those leaves inoculated with the challenge strain RNA. No accumulation of challenge ds RNAs or virions occurred in later formed leaves. Tomato and tobacco plants infected with CMV-S were not protected from infection by tobacco mosaic virus.     

Enzyme cytochemical identification of single-stranded and double-stranded RNAs in virus-infected plant and insect cells

A method is described for identifying and locating single-stranded (ss) and double-stranded (ds) RNAs by electron microscopy in plant and insect cells. Single-stranded RNA in aldehyde-fixed tissues is identified by its susceptibility to pancreatic ribonuclease when incubated in media containing both low or high salt concentrations, and dsRNA is identified by its susceptibility to the enzyme in media of low but not high salt concentrations. The validity of this method for identification of intracellular dsRNA has been verified with plant and insect cells infected with Fiji disease and maize wallaby ear viruses, respectively, both of which contain dsRNA genomes. The method has also been used for the identification of dsRNA in vesicles of virus-induced inclusions in plant cells infected with Echtes Ackerbohnenmosaikvirus, a member of the comovirus group which contains an ssRNA genome. The need for critical interpretation of observations of structures in RNase-treated cells is discussed.     

In vitro assembly of poliovirus: V. Evidence that the self-assembly activity of 14 S particles is independent of extract assembly factor(s) and host proteins

Experiments were performed to determine whether or not the self-assembly of 14 S precursor particles into empty capsids was caused by the contamination of certain 14 S isolates with assembly factor(s) present in poliovirus-infected cell extracts. The self-assembly capacity of 14 S particle preparations was found to be directly related to the amount of viral-specific protein present. Dilution of 14 S particles markedly inhibited their self-assembly activity, whereas using ultrafiltration methods to concentrate 14 S particles increased their self-assembly activity. No consistent relationship was found for the presence or absence of particular viral or host proteins and the ability of 14 S particle preparations to self-assemble. The self-assembly capacity of 14 S particles was more sensitive to uv-inactivation than their ability to assemble into empty capsids in the presence of infec cted cell extracts. On the basis of these data and a detailed reanalysis of the effect of relative initial 14 S particle concentration on the rate of formation of empty capsids in extracts, we propose that the assembly of 14 S particles into empty capsids occurs in two steps, an initiation event and subsequent polymerization, and that extracts act by promoting the initiation event.     

Protein analysis of defective interfering lymphocytic choriomeningitis virus and persistently infected cells.

Defective interfering (DI) lymphocytic choriomeningitis virus (LCMV) was purified from the culture fluids of BHK 21/13S and L-929 cells persistently infected with LCMV. DI LCMV sedimented in renografin-76 gradients to a density slightly less than standard (S) LCMV (1.13 vs 1.14 g/cm³). Polyacrylamide gel electrophoretic analysis of [³⁵S]methionine-labeled DI virus revealed a major 63,000-dalton polypeptide corresponding to the S virion nucleoprotein (NP), and two minor polypeptides corresponding to the S virion 54,000? and 35,000-dalton glycopeptides. No differences in polypeptide composition were detected between the DI and S virions. Exposure of cells to DI virus before S virus challenge inhibited the intracellular synthesis of the NP. Cells persistently infected with LCMV released no detectable S virus or temperature-sensitive mutants but did release DI LCMV. The production of DI LCMV by these cultures was 10? to 100-fold lower than S virus production during acute infections. These persistently infected cells contained intracytoplasmic NP, detectable by immunofluorescence, but its rate of synthesis was too low to be detected by the radiolabeling methods used. Although present in the cytoplasms, detectable viral antigens were absent from the cell membranes of many of these persistently infected cells. Thus, cells persistently infected with LCMV produce relatively low levels of DI virus which can inhibit viral protein synthesis. These factors may act to render infected cells resistant to immunosurveillance mechanisms during persistent infections in vivo.     

Acute infection of differentiated neuroblastoma cells by latency-positive and latency-negative herpes simplex virus ts mutants.

Latent herpes simplex virus (HSV) appears to be selectively harbored in neurons. In initial attempts to understand this unique relationship, we have studied viral-neuronal interaction in vitro utilizing mouse C1300 neuroblastoma cells and selected latency-positive and latency-negative temperature-sensitive virus mutants. Comparative studies were made in baby hamster kidney (BHK) cells (where the mutants were first characterized) and, where possible, in mouse brain neurons in situ. Neurons in situ and neuroblastoma cells were productively infected by wild-type virus, the mutants were restricted at the non-permissive temperature, and DNA phenotypes in neuroblastoma cells were identical to those found in BHK cells. However, two mutants were found to have significantly different ultrastructural phenotypes at the restrictive temperature when neuronal infections were compared to BHK infections. In addition, the wild-type virus induced increased amounts of several polypeptides in neuroblastoma cells, and the processing of immediate-early polypeptides was impaired in infections with two mutants. These observations indicate that compared to BHK infections, neuronal infections do exhibit unique characteristics. Finally, the results are discussed with respect to both the general nature of latent herpetic infections and to the viral-specific information involved in establishment of these infections.     

Appearance of defective simian virus 40 following infection of cultured human glioblastoma cells.

Infection of human glioblastoma cells (A172) with SV40 is followed by virus propagation and cell death. Abbreviated viral genomes, however, appear rapidly in the virus pool. These are shown to be defective (i.e., they require helper activity from standard virus) and to exhibit interference with the lytic growth of standard virus. Experiments with triply plaque-purified SV40 have demonstrated (1) that substantial levels of defectives appear within one very low multiplicity infection of A172 cells or within two to three moderately low multiplicity passages; (2) that defectives are in fact generated at a high rate in A172 cells; and (3) that the generation and amplification processes do not select strongly for particular genome sizes. Carrier cultures have been established in the A172 cells. The unusual features of the accumulation of defective SV40 in A172 cells are the high rate of accumulation and the lack of a requirement for high multiplicity passage to amplify and to maintain high levels of defectives.     

Observations on the membrane organization of standard and incomplete influenza grown in MDBK cells.

The phospholipid content and composition of incomplete WSN influenza virions grown in MDBK cells were compared with standard particles grown at low multiplicity of infection and found to be indistinguishable. The ESR spectra of incomplete and standard virions labeled with a stearic acid spin label were also indistinguishable, indicating that the rigidity of the viral bilayer was not measurably different in the two kinds of particles. Evidence is presented that a small peptide or peptides of the viral glycoproteins remains associated with the particle after proteolytic digestion of the glycoprotein spikes. Since the membranes of incomplete viral particles have previously been shown to contain approximately twice the amount of glycoproteins present in standard virus, it is concluded that these small peptides exert no measurable effect on bilayer composition or rigidity.     

Interferon-induced translation defects in a cell-free protein-synthesizing system from mouse erythroleukemia cells

The mechanism of the interferon-induced translation inhibition was studied in vitro using a fractionated cell-free protein-synthesizing system prepared from Friend virus-transformed mouse erythroleukemia cells and L cells. The soluble fraction of the system was prepared from nonpreincubated S30 lysates. To lower endogenous protein synthesis, the ribosomes were prepared from extracts treated with micrococcal nuclease at 20°. If the components were prepared from interferon-treated cells, viral and cellular mRNA translation was inhibited. In the cell-free system prepared from interferon-treated mouse erythroleukemia cells, the translation defect could be localized in the high-speed supernatant, while ribosomes from interferon-treated and control cells were of comparable activity. In contrast, ribosomes from interferon-treated L cells prepared by the same method were unable to support translation of exogenous mRNAs. The interferon-induced translational defect in either cell-free system could not be overcome by adding tRNA. Aminoacylation and stability of endogenous leucine-specific tRNAs was not impaired in the high-speed supernatant of both of the cell-free systems from interferon-treated cells.     

Temperature-sensitive mutants of influenza virus XVI. Transfer of the two ts lesions present in the Udorn/72-ts- 1 A2 donor virus to the Victoria/3/75 wild-type virus

Previously, the influenza A/Hong Kong/68-ts-1[E] virus and its recombinants (38° shutoff temperature of plaque formation) were shown to be insufficiently attenuated for persons who lacked immunity to both the hemagglutinin and neuraminidase surface glycoproteins, i.e., doubly seronegative individuals. To meet the need for immunization of such individuals, a virus more defective than the ts-1[E] recombinants was produced. The resulting virus, designated Udorn/72-ts-1A2, possessed is mutations in genes represented by complementation-recombination groups 1 and 5 and was more restricted in replication in vitro at 37° and in vivo in the hamster's lungs and nasal turbinates than the Udorn/72ts-1[E] virus. These properties suggested that the Udorn/72 ts-1A2 virus might serve as a donor of its two ts lesions to new variants of influenza A virus to produce attenuated strains for use in doubly seronegative individuals. The Udorn/72-ts-1A2 virus was mated with the influenza A/Victoria/3/75 wild-type virus, and six is recombinant viruses bearing the Vic/75 hemagglutinin were isolated. Five of these Vic/75-ts-1A2 recombinants had a 37° shutoff temperature and two ts lesions like their Udorn/72-s-1A2 parent. Each of these clones replicated to low titer or not at all in the hamster's lungs and were 100-fold restricted in the nasal turbinates like their ts parental virus. Each of 158 isolates recovered from hamsters infected with the parental Udorn/72-ts-1A2 virus or one of its Vic/75 ts double lesion recombinants retained the ts phenotype. One Vic/75-ts-1A2 recombinant clone that had a 38°hutoff temperature, and only the group 1 ts lesion replicated in the hamster's lungs to a level intermediate between that of the parental ts-1A2 and wild-type virus. All isolates recovered from hamsters infected with this recombinant were also ts. Infection of hamsters with a Vic/75-ts-1A2 recombinant, bearing the group 1 and group 5 ts mutations, induced resistance to homologous wild-type virus challenge. Since the parental and double lesion recombinant ts-1A2 viruses had similar restriction of replication in vitro at 37° and in vivo, it is likely that the two ts-1A2 is lesions will effect a similar level of attenuation following transfer into other antigenic variants of influenza A virus.     

Biochemical evidence of the nonintegrated status of Marek's disease virus DNA in virus-transformed lymphoblastoid cells of chicken.

The status of latent virus genomes in MKT-1 cells, a lymphoblastoid cell line derived from a kidney tumor in a Marek's disease virus (MDV)-infected chicken, has been studied. The cells are virus nonproductive and contain 15 virus genomes per cell, the majority of which can be separated from high molecular weight cell DNA. The fast-sedimenting virus DNA from MKT-1 cells showed an S value of 100 S in a neutral gradient and 200 S in an alkaline gradient by centrifugation and banded at the position of SV40 component 1 DNA in ethidium bromide-cesium chloride equilibrium centrifugation. The data indicate that the 100 S molecule has all the characteristics of closed circular DNA. Thus, the latent MDV DNA in MKT-1 cells is another example of the circular plasmid form of latent herpesvirus DNA. The possibility of integration of a small portion of the virus genomes has not been determined.     

Selection of a mutant S1 gene during reovirus persistent infection of L cells: role in maintenance of the persistent state

LR-7 cells, variant L cells derived from a type 3 reovirus persistently infected (p.i.) carrier culture (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25, 325-332, 1983) were used to define the viral genes critical for maintenance of the persistent state. A cloned viral isolate (L/C virus) derived from the p.i. culture replicated normally in LR-7 cells, while wild-type (wt) viruses of the three reovirus serotypes replicated less efficiently. To identify the viral gene(s) permitting enhanced replication of L/C virus in LR-7 cells, viral reassortants were prepared by mixed infection of L cells with L/C virus and type 1 wt. Study of the one-step growth curves and final yields of large numbers of reassortants in both L cells and LR-7 cells revealed that the presence of the S1 gene from L/C virus was critical for normal viral replication in LR-7 cells. However, this phenotype was suppressed by the simultaneous presence in reassortants of both the M2 and S4 genes from the type 1 wt parent. The critical change in the S1 gene occurred by passage 13 (63 days) after initiation of the carrier culture. Although multiple mutations are present in the viral population from p.i. cultures, certain specific mutations can be identified as critical for maintenance of the persistent state.     

Properties of the multicapsid virions of murine cytomegalovirus.

φ6 is a bacteriophage containing a genome of three pieces of double-stranded RNA and an envelope composed of lipids and proteins. The particles formed during infection of a nonsuppressor host by wild-type virus and six classes of nonsense mutants were investigated in this study.Mutants defective in the synthesis of P5 and P1l formed particles with RNA and lipid and all virion proteins but P5 and P11. Mutants defective in the synthesis of P3 made particles with lipid and RNA but also lacked P6. Particles formed by mutants defective in P6 were similar and lacked P3. These particles do not adsorb to host cells, indicating a role for P3 and P6 in attachment.Particles formed by a mutant defective in the synthesis of P9, P5, and P11 contained RNA but no lipid and also lacked P10, P3, and P6, the other membrane proteins. Particles formed by a mutant unable to form P12, a nonstructural protein, also lacked P9, P10, P3, P6, P5, and P11, and contained no lipid.We conclude that the core of the virion can be formed without the membranous exterior and that membrane protein P9 and the nonstructural protein P12 are necessary for the formation of the lipid-containing structure.